The goal of this project is to characterize the signaling pathways that regulate synaptogenesis in the developing brain. Previous studies have demonstrated that synapses differentiate in a series of steps, one of which is the clustering of postsynaptic neurotransmitter receptors. In the neuromuscular junction, clustering of the cholinergic receptors is induced by a proteoglycan (agrin), which activates a receptor tyrosine kinase (MuSK). In the central nervous system, it is not clear what regulates the clustering of postsynaptic receptors such as the NMDA receptors at excitatory synapses. Preliminary data from the Sponsor's laboratory indicate that activation of Eph receptor tyrosine kinases on cultured hippocampal neurons induces the clustering of a NMDA receptor subunit, NR1. Furthermore, Eph receptor ligands (ephrins) induce a biochemical interaction between the Eph and NMDA receptors. This study will examine the protein-protein interactions that mediate ephrin-induced NMDA receptor clustering through three specific aims. First, we will identify the domains of Eph and NR1 receptors that are critical for ephrin-induced receptor clustering. Then, we will investigate the role of tyrosine phosphorylation on the clustering of NMDA receptors by Eph tyrosine kinases. Finally, the cellular mechanisms of NMDA receptor clustering will be examined through two sets of experiments. We will present the ephrin ligands on the cell membrane and determine their effect on the NMDA receptors on neighboring neurons. Mechanisms of synapse formation are of particular interest to pediatric neurologists because aberrant synapse formation is likely to underlie common developmental disorders such as mental retardation and epilepsy. Through intensive training in basic science under the supervision of Dr. Michael Greenberg and clinical practice in pediatric neurology at the Children's Hospital in Boston, the candidate expects to become a physician- scientist equipped with the molecular and cellular tools to study neuronal dysgenesis in children.